In this paper we present the analysis of nucleolar organizer regions (NORs) in five species of the order Pleuronectiformes (Scophthalmus maximus, Scophthalmus rhombus, Platichthys flesus, Solea solea and Solea lascaris), a group of fish poorly studied cytogenetically. In spite of the small chromosome sizes, which characterize the karyotypes of this group, we implement both classical (Ag- and CMA3-staining) and molecular (in situ hybridization with a major rDNA probe) techniques for NOR location. NORs were localized in a single chromosome pair in all species studied and showed size variation within each species. Evidence of a NOR-site polymorphism was detected in Platichthys flesus and Scophthalmus maximus. NOR location was apparently the same within families (S. rhombus vs. S maximus, and S. solea vs. S. lascaris) but clearly differed among families, and supports the phylogenetic relationships proposed for the order Pleuronectiformes.